The discovery of electrophoretic or chromatographically fast moving minor hemoglobin components of blood was first accomplished by Schroeder et al, J. Am. Chem. Soc., 80:1628 (1958). Various other investigators verified these findings and identified these fast fractions as carbohydrate-linked modifications of normal adult hemoglobin designated as Hgb A.sub.1 a, A.sub.1 b, and A.sub.1 c; see, for example, Clegg et al, ibid., 81:6065 (1959).
Studies have shown that all three fractions are post-transitional modifications and are formed slowly and continuously throughout the 120-day life span of the erythrocyte.
Structural analysis of Hgb A.sub.1 c shows a glucose moiety at the amino terminus of each beta chain of normal adult hemoglobin. The formed red cell is unable to initiate protein synthesis. Hgb A.sub.1 c is therefore a post formation product, the biosynthesis of which is achieved by the non-enzymatic production of a Schiff base between the aldehyde of the carbohydrate and the amino terminus. An Amadori rearrangement to a stable ketamine then occurs. Hemoglobins A.sub.1 a and A.sub.1 b appear also to be adducts of carbohydrate metabolites found in normal human erythrocytes. However, their mode of formation is not yet as clearly understood.
Rahbar, in 1968, first reported the presence of elevated levels of glycosylated hemoglobins (glycohemoglobins) in diabetic patients; Clin. Chim. Acta, 22:296 (1968). It was also later shown that these increased levels did not represent the effects of complications of the disease. Finally, hospitalized diabetics showed a marked reduction in Hgb A.sub.1 c as control of the disease was accomplished; see Lancet, 2:734 (1977); N. Eng. J. Med., 95:417 (1976).
Hemoglobin A.sub.1 c is the most abundant of these glycohemoglobins comprising about 3-6% of total hemoglobin concentration. Unlike the lesser components A.sub.1 a and A.sub.1 b, its concentration is in direct proportion to the average amount of circulating blood glucose. It has also been demonstrated that the levels are highest in the oldest of the population of red cells in both diabetics and normal subjects. The measurement of glycohemoglobins of which Hgb A.sub.1 c is the major component, is, therefore, a reflection of the glucose levels of a patient over a long period of time and yields a clear assessment of control status with relation to therapy.
Until the present invention, the measurement of Hgb A.sub.1 c was a research oriented procedure requiring both sophisticated equipment and considerable time and expense.
Accordingly, one object of the invention is to provide a new method for determining the content or percentage of glycohemoglobins relative to total circulating hemoglobins in patient whole blood.
Another object of the present invention is to provide a new method for determining the percent of glycohemoglobin as an aid to the diagnosis and monitoring of the diabetic patient.
Other objects, features and advantages of the present invention will become apparent from the subsequent description and appended claims.